Ion channels are cellular proteins that regulate the flow of ions, including calcium, potassium, sodium and chloride, into and out of cells. These channels are present in all human cells and affect such processes as nerve transmission, muscle contraction and cellular secretion. Among the ion channels, potassium channels are the most ubiquitous and diverse, being found in a variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels have now been associated with a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Moreover, studies have indicated that K+ channels are a therapeutic target in the treatment of a number of diseases including central or peripheral nervous system disorders (e.g., migraine, ataxia, Parkinson's disease, bipolar disorders, trigeminal neuralgia, spasticity, mood disorders, brain tumors, psychotic disorders, myokymia, seizures, epilepsy, hearing and vision loss, psychosis, anxiety, depression, dementia, memory and attention deficits, Alzheimer's disease, age-related memory loss, learning deficiencies, anxiety, traumatic brain injury, dysmenorrhea, narcolepsy and motor neuron diseases), as well as targets for neuroprotective agents (e.g., to prevent stroke and the like). The compounds of the invention are also useful in treating disease states such as gastroesophogeal reflux disorder and gastrointestinal hypomotility disorders, irritable bowel syndrome, secretory diarrhea, asthma, cystic fibrosis, chronic obstructive pulmonary disease and rhinorrhea, convulsions, vascular spasms, coronary artery spasms, renal disorders, polycystic kidney disease, bladder spasms, urinary incontinence, bladder outflow obstruction, ischemia, cerebral ischemia, ischemic heart disease, angina pectoris, coronary heart disease, Reynaud's disease, intermittent claudication, Sjorgren's syndrome, arrhythmia, hypertension, myotonic muscle dystrophia, xerostomi, diabetes type II, hyperinsulinemia, premature labor, baldness, cancer, and immune suppression.
Potassium channels are made by alpha subunits that fall into at least 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7): 805–829 (1997)). Three of these families (Kv, eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25): 14066–14071 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273: 3509–3516 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing two transmembrane domains, and an eighth functionally diverse family (TP, or “two-pore”) contains two tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493: 625–633 (1996); Shi et al., Neuron 16(4): 843–852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384: 80–83 (1996)).
Slo or BK potassium channels are large conductance potassium channels found in a wide variety of tissues, both in the central nervous system and periphery. They play a key role in the regulation of processes such as neuronal integration, muscular contraction and hormone secretion. They may also be involved in processes such as lymphocyte differentiation and cell proliferation, spermatocyte differentiation and sperm motility. Three alpha subunits of the Slo family have been cloned, i.e., Slo1, Slo2, and Slo3 (Butler et al., Science 261: 221–224 (1993); Schreiber et al., J. Biol. Chem., 273: 3509–16 (1998); and Joiner et al., Nature Neurosci. 1: 462–469 (1998)). These Slo family members have been shown to be voltage and/or calcium gated, and/or regulated by intracellular pH.
Certain members of the Kv family of potassium channels were recently renamed (see, Biervert, et al., Science 279: 403–406 (1998)). KvLQT1 was re-named KCNQ1, and the KvLQT1-related channels (KvLR1 and KvLR2) were renamed KCNQ2 and KCNQ3, respectively. More recently, a fourth member of the KCNQ subfamily was identified (KCNQ4) as a channel expressed in sensory outer hair cells (Kubisch, et al., Cell 96(3): 437–446 (1999)).
SK channels are small conductance, Ca2+-activated K+ channels that underlie neuronal slow afterhyperpolarization and mediate spike frequency adaptation (Khawaled et al., Pflugers Arch. 438: 314–321 (1999)). SK channels are present in many central neurons and ganglia, where their primary function is to hyperpolarize nerve cells following one or several action potentials, in order to prevent the occurrence of long trains of epileptogenic activity. The SK channels are also present in several peripheral cells including skeletal muscle, gland cells, liver cells, and T-lymphocytes. The significance of SK channels in normal skeletal muscle is not clear, but their number is significantly increased in denervated muscle, and the large number of SK channels in the muscle of patients with myotonic muscle dystrophia suggests a role in the pathogenesis of the disease.
Three SK channels have been identified to date: SK1, SK2 and SK3 (Rimini et al., Brain Res. Mol. Brain Res. 85: 218–220 (2000)). The quantities of SK1, SK2 and SK3 expression in human brain have been measured using TaqMan RT-PCR on a range of human brain and peripheral tissue samples. SK1 expression was found to be restricted to the brain whereas SK2 and SK3 are more widely expressed.
SK channels have been shown to have a distinct pharmacological profile. For example, using patch clamp techniques, the effects on SK2 subtype channels of eight clinically relevant psychoactive compounds structurally related to the tricyclic antidepressants were investigated (Dreixler et al., Eur. J. Pharmacol. 401: 1–7 (2000)). The compounds evaluated included amitriptyline, carbamazepine, chlorpromazine, cyproheptadine, imipramine, tacrine and trifluperazine. Each of the compounds tested was found to block SK2 channel currents with micromolar affinity. In contrast, the cognitive enhancer linopirdine was ineffective at inhibiting SK channels. A number of neuromuscular inhibiting agents which affect SK channels exist, e.g. apamin, atracurium, pancuronium and tubocurarine (Shah et al., Br J Pharmacol 129: 627–30 (2000)).
Patch clamp techniques have been used to study the effect of the centrally acting muscle relaxant chlorzoxazone and three structurally related compounds, 1-ethyl-2-benzimidazolinone (1-EBIO), zoxazolamine, and 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 channels) expressed in HEK293 mammalian cells (Cao et al., J Pharmacol Exp. Ther. 296: 683–689 (2001)). When applied externally, chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca2+-free intracellular solution.
The effects of metal cations on the activation of recombinant human SK4 (also known as hIK1 or hKCa4) channels has also been studied (Cao and Houamed, FEBS Lett. 446: 137–141 (1999)). The ion channels were expressed in HEK 293 cells, and tested using patch clamp recording. Of the nine metals tested, cobalt, iron, magnesium, and zinc did not activate the SK4 channels when applied, at concentrations up to 100 μM, to the inside of SK4 channel-expressing membrane patches. Barium, cadmium, calcium, lead, and strontium activated SK4 channels in a concentration-dependent manner. The rank order of potency was at Ca2+>Pb2+>Cd2+>Sr2+>Ba2+.
WO 97/48705 discloses a particular group of chemical compounds useful as calcium activated potassium channel inhibiting agents. U.S. Pat. No. 5,739,127 and U.S. Pat. No. 5,760,230 discloses a series of 2,4′-bridged bis-2,4-diaminoquinazolines having activity towards apamine-sensitive potassium channels. None of the aforementioned references disclose that the compounds set forth therein exhibit any selectivity towards the SK channel.
WO 00/01676 discloses a genus of potassium channel inhibiting agents based on an amine-substituted bis-benzimidazole scaffold in which the amine-substituted benzimidazole moieties are joined by a linker arm. Similar compounds are set forth in U.S. Pat. No. 5,922,794. Each of the disclosed bis-benzimidazoles is derivatized at one or more of the endocyclic imidazole nitrogen atoms Moreover, the majority of the disclosed bis-benzimidazole compounds include a linker arm that is attached to either an endocyclic amine moiety or to the amine substituent of the amine-substituted benzimidazole subunits.
In contrast to the compounds set forth in WO 00/01676, and U.S. Pat. No. 5,922,794, the present invention provides a genus of SK channel modulators that are based on a bis-benzimidazole scaffold in which the benzimidazole moieties are linked through the carbon atom at position-2 of the imidazole ring system. The compounds of the invention are potent and specific modulators of SK channels.